One aspect of this project relates to determining the role of the ubiquitin system in mitochondrial protein turnover including membrane dynamics and how this in turn relates to cellular energetics in cancer and to apoptosis. Related to this is our interest in understanding mitochondrial protein quality control and how the ubiquitin system is involved in this. This project was begun in yeast, had expanded to mammalian systems, and now moved back to yeast for initiation of quality control studies. We have determined in yeast that the E3 SCF-Mdm30 plays a critical role in targeting the mitofusin, Fzo1, for degradation and that this degradation is integral to the process of mitochondrial outer membrane fusion. In mammals we have established a pathway leading from cell stress to activation of JNK, which leads to phosphorylation of mammalian mitofusin 2 (Mfn2). This in turn leads to the recruitment of the large HECT domain ubiquitin ligase Huwe1. Mfn2 is then ubiquitinated and degraded by the proteasome leading to mitochondrial fragmentation and cell death. Thus we have established a new pathway from cell stress to degradation of a specific mitochondrial protein to apoptosis. Ongoing work on Mfn2 relates to understanding the role of other ubiquitin ligases in regulating Mfn2 degradation. Our ongoing quality control studies in mitochondria use temperature sensitive alleles of yeast proteins and assess the requirements for their degradation. This work, to date, has resulted in the discovery of proteins of the ubiquitin system that were not previously thought to be involved in degradation of mitochondrial proteins. It has also led us in new and completely unexpected directions regarding the role of the ubiquitin system in regulating plasmid segregation. A major aspect of this project relates to degradation from the endoplasmic reticulum (endoplasmic reticulum (ER)-associated degradation; ERAD). Together with collaborators we are studying E2s and E3s critical to this process. gp78/RNF45, also known as the tumor autocrine motility factor receptor (AMFR), was discovered by our laboratory to be an ubiquitin ligase resident to the ER. We have determined that gp78 plays essential roles in the degradation of multiple substrates functioning together with an E2 that we first characterized, originally known as MmUBC7 and now referred to as Ube2G2. We have determined that multiple domains within gp78 function together to mediate its ubiquitin ligase activity. These include its RING finger, an ubiquitin-binding Cue domain and a novel region that specifically recruits Ube2g2 independent of the RING finger, referred to as the G2BR. Moreover, we now know that expressing the G2BR in isolation can block ERAD and induce ER stress. We are exploring the potential for such expression or other means of blocking the interaction between the gp78 and Ube2g2 as a means to target cells that are predisposed to ER stress, such as multiple myeloma cells, to undergo apoptosis. gp78 levels are correlated with the metastatic potential of tumors including melanomas and lung cancers. In vivo studies in mice using knockdowns of gp78 and re-expression in cells, in xenograft models, have now determined that gp78 does, in fact, play an important role in the metastatic potential of multiple different sarcomas and that this potential for metastasis requires intact ubiquitin ligase function of this protein. We have also determined that gp78 targets the metastasis suppressor, KAI1 (CD82) for degradation in sarcomas. This provides at least a partial explanation for our findings. We have generated mouse models to study gp78, studies are underway to determine the role of this ubiquitin ligase in breast cancer including in the metastasis of spontaneously arising tumors. gp78 has been reported in the literature as being responsible for the targeting of the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, for degradation. This was an important finding in considering approaches to regulation of sterols. However, we have found, through a thorough analysis of multiple different cell lines, that this observation is an artifact. On the other hand, gp78 does target Insig-1, a major negative regulator of sterol biosynthesis, for degradation. This now makes gp78 an attractive target, and an alternative to statins, for treatment of hypercholesterolemia. gp78 represents a potentially important target in both cancer and metastasis. Studies in mouse models are ongoing to evaluate the in vivo significance of these findings. At the same time, we are exploring the potential for identifying deubiquitinating enzymes involved in regulating critical regulators of cholesterol and fatty acid biosynthesis in the endoplasmic reticulum and Golgi. These represent potentially important targets, and may potentially be more druggable then ligases.